1. Field of the Invention
This invention relates to the combined use of calibrated fluorescent biological cells with calibrated fluorescent microbeads to compensate for different responses of different flow cytometers.
2. Description of the Related Art
Flow cytometers are used to analyze biological cells and particles in a fluid sample by intersecting a thin stream of the fluid by an illumination source, usually a laser beam. The resulting forward and right angle scattered and fluorescent light is analyzed with photomultiplier tubes (PMTs). The fluorescence channels of a flow cytometer, designated by F11, F12, F13, etc., are each set with barrier filters to detect a selected specific dye while filtering out signals from other wavelengths.
U.S. Pat. Nos. 4,714,682, 4,767,206, and 4,774,189, and U.K. Patent 2,172,104 describe calibration of a flow cytometer using highly uniform microbeads which have excitation and emission spectra that match that of the unknown samples, as well as describing the synthesis and composition of said highly uniform microbeads. Matching spectra of microbeads and cells in this way allows direct comparison of data among flow cytometers which have different barrier filters so long as the sample and the calibration microbeads are analyzed under the same instrument conditions and settings. The totality of these patents and all other patents and any other publications cited herein and/or referred to in the Cross-Reference to Related Applications is hereby incorporated herein by reference.
Fixed calf thymocyte nuclei stained with fluorescent dyes, e.g., fluorescein and phycoerythrin, were available in the public domain as samples between 1983 and 1987 under the trade name, Fluorotrol.TM. by Ortho Diagnostics Instrument Division (now part of Becton Dickinson Immunodiagnostic Systems). Fluorotrol.TM. thymocytes nuclei consisted of three different populations of fixed calf thymocytes nuclei: an unstained, a "dim" and a "bright" population. Fluorotrol.TM. was not designed as a calibration agent, but rather a reference material because the coefficient of variation on the fluorescence intensity of these cells was relatively wide, e.g., 10-20%, however they did possess similar size, refractive indices, and spectral properties as cellular samples normally analyzed with flow cytometers. References: Brown M, Hoffman R, Kirchanski S: Controls for flow cytometers in hematology and cellular immunology. Ann. NY. Acad. Sci. 468: 93-103, 1986; and Vogt, Cross, Phillips and Henderson (1989). A model System Evaluating Fluorescein-Labeled Microbeads as Internal Standards to Calibrate Fluorescence Intensity on Flow Cytometers. Cytometry 10: 294-302.
Similarly, when the size of cells is measured via forward light scatter with a flow cytometer which has been calibrated by relatively high refractive index calibration microbeads, the cells are found to be smaller than when measured by other means, e.g., light or scanning electron microscopy.
Although previously available microbeads have enabled instrument calibration, fluorescence intensity measurements of stained cells (e.g., lymphocytes stained with fluorochromes specific for CD4, a common lymphocyte surface marker) reveal that there is a large coefficient of variation (22.7%) when the cells are measured on different flow cytometers (see Example 4). A number of factors other than spectral matching appear to contribute to this imprecision, e.g., variation in excitation energy, geometry and material of the fluidics, and differences in the refractive index and scatter properties between the calibration microbeads and the cell samples.
One of the major sources of imprecision is due to the differences in the influence of excitation energy (i.e., laser power) on the fluorescence intensity (FI) of the calibration microbeads and the cell samples. As laser power increases, the FI of the microbeads becomes proportionally higher than the FI of the cell samples (see FIG. 8). This difference is probably related to the relatively low refractive index of cells (since a large component of their mass is water) and other factors which allows slightly more efficient excitation of the cells compared to the microbeads. This effect influences calibration of cell samples by causing a shift to lower MESF values (FIG. 9) (MESF units are measures of FI expressed as equivalent molecules of soluble fluorochrome that give the same FI as the cells or microbeads).
This difference may be seen from a comparison of FIGS. 1 and 2 which show illumination of microbeads with lower and higher laser beams, respectively, which is projected toward the bead from the left of the bead in the Figures. The smaller portion of bead illuminated with a low power laser beam results in a shift to lower numbers of MESF units in the channels.
These data suggested that some factors are required to account and correct for the differences and imprecision of fluorescence intensity and size measurements via flow cytometry.
It is therefore an object of this invention to provide cells and microbeads which are calibrated which may be used to adjust the calibration of flow cytometers by correcting the fluorescence intensity calibration plot of the flow cytometer.
It is a further object of this invention to provide a method of standardizing flow cytometers by correcting the fluorescence intensity calibration plot using calibrated cells and microbeads.
It is a further object of the invention to provide a method of increasing accuracy of size measurements using calibrated microbeads and calibrated cells.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.